HIV vaccine development has been hampered by unprecedented challenges, mostly related to the unique properties of the viral envelope trimer, a cleverly engineered entry machinery that features an extraordinary assortment of immune-evasion tactics, including antigenic variation, conformational camouflage and heavy glycosylation of exposed surfaces. Further insights into the complex structure of the HIV-1 envelope and its protective shield will be critical to guide the design of a protective vaccine. 1) Two tyrosine residues in the V2 domain of HIV-1 gp120 can be modified by sulfation. Tyrosine sulfation is a post-translational modification that occurs in approximately 7% of mammalian proteins, including several host proteins that interact with HIV-1 gp120 such as the coreceptors CCR5 and CXCR4, and monoclonal antibodies to the coreceptor-binding site (eg, 412d) and the V2 loop (eg, PG9). We noticed that the central region of V2 contains two tyrosine residues (Tyr173 and Tyr177) that are identically spaced as two critical sulfotyrosines (Tys) in the N-terminus of CCR5 (Tys10 and Tys14). Using different experimental approaches, we demonstrated that the two tyrosines in V2 are indeed sulfated. The level of V2 tyrosine sulfation is generally low when gp120 is expressed in continuous cell lines due to low endogenous levels of tyrosil-protein sulfo-transferase (TPST) activity; however, sulfation can be enhanced by TPST overexpression. Corroborating the biological relevance of this observation, we consistently detected high levels of gp120 sulfation in HIV-1 isolates grown in primary human T cells, which are the primary targets for HIV replication in vivo. These results document the presence of sulfated tyrosines within a viral envelope protein, which thus far had been reported only for the varicella-zoster herpesvirus. 2) The tyrosine-sulfated region of gp120 is a functional mimic of the CCR5 N-terminal domain and mediates intramolecular interaction with the CCR5-binding site at the base of the V3 loop To elucidate the functional role of the tyrosine-sulfated region of V2, we made the following observations: i) a tyrosine-sulfated peptide mimetic derived from V2, but not its unsulfated counterpart, interacts with the CCR5-binding site at the base of V3, suggesting that this V2 region mediates intramolecular interaction between V2 and V3 in the pre-fusion envelope trimer. This model was confirmed by plasmon resonance studies with both monomeric and trimeric gp120 and is compatible with the position of the V2 tyrosines in the recent crystal structure of a soluble cleaved trimer (BG505 SOSIP.664); ii) the sulfated region of V2 acts as a functional mimic of the sulfated N-terminal domain of CCR5, which interacts with the base of V3 after gp120 activation with CD4; iii) sulfation of the V2 tyrosines, especially of Tys177, provides the largest fraction of the V2-V3 binding energy; iv) NMR studies confirmed the CD4-dependent interaction of the sulfated V2 peptide with gp120, which is specifically inhibited by a CCR5 N-terminal sulfated peptide.Moreover, the bound V2 peptide adopts a helical configuration which is analogous to that documented for a CCR5-derived tyrosine-sulfated N-terminal peptide bound to gp120-CD4 complexes. 3) Tyrosine sulfation of V2 stabilizes the closed, pre-fusion conformation of the HIV-1 envelope trimer and thereby modulates HIV-1 neutralization sensitivity To gain additional insights into the role of V2 tyrosine sulfation, we tested the effects of modulating the levels of sulfation using either TPST2 overexpression or treatment with the sulfation inhibitor, sodium chlorate. We demonstrated that tyrosine sulfation modulates the antigenic profile of gp120 by shifting the conformational equilibrium toward the closed, antibody-protected conformation. In fact, enhancement of sulfation reduces the accessibility of the CD4- and coreceptor-binding sites, while inhibition of sulfation has reciprocal effects, favoring the adoption of a more open trimer conformation. Of note, antibodies that preferentially recognize the closed trimer, such as the glycan-dependent anti-V2 antibodies PG9 and PG16, exhibit a reversed pattern with increased binding to the fully sulfated envelope and decreased recognition of the chlorate-treated trimer. Strikingly, the pattern of HIV-1 neutralization sensitivity under conditions of increased or decreased tyrosine sulfation precisely mirrors the antigenic profiles. Analogous results were obtained using alanine mutants of the V2 sulfated tyrosines, confirming the key role of such tyrosines in modulating gp120 epitope accessibility and HIV-1 neutralization. Altogether, these results identify the sulfotyrosine-mediated V2-V3 interaction as a key structural constraint that contributes to stabilizing the native, antibody-protected conformation of the HIV-1 envelope trimer. 4) Tyrosine sulfation of V2 is a mechanism of immune evasion To investigate whether the sulfotyrosine-mediated V2-V3 interaction could promote HIV-1 immune evasion, we tested the neutralizing capacity of a large panel of sera from HIV-infected patients against HIV-1 pseudotypes carrying either the wild-type or the doubly alanine-substituted (Y173A/Y177A) mutant BaL envelope. The results were dramatic, showing a staggering increase in neutralization sensitivity upon mutation of the two V2 tyrosines, with half-maximal neutralizing titers reaching beyond 1:50,000 for the majority of the sera tested, while the wild-type virus was typically neutralized within the 1:10-1:500 dilution range. These data demonstrate that the V2 tyrosines help to maintain the envelope trimer in its antibody-protected pre-fusion conformation, preventing the binding of abundantly produced host antibodies directed against critical neutralization epitopes such as the CD4- and coreceptor-binding sites. 5) Tyrosine-sulfated peptides derived from the V2 loop are potent and specific inhibitors of HIV-1 entry To further investigate the structural and functional relevance of tyrosine sulfation in the gp120 V2 loop, we derived short mimetic peptides from the sulfated region of V2 and tested their biological activity. We found that a doubly-sulfated peptide, but not its unsulfated counterpart, binds with nanomolar affinity to gp120 when the glycoprotein is pre-activated with soluble CD4 (sCD4); furthermore, it inhibits binding of a sCD4-treated soluble cleaved gp140 trimer (BG505 SOSIP.664) to CCR5. Accordingly, the peptide specifically blocks HIV-1 entry and fusion by preventing coreceptor utilization. Experiments with singly-sulfated peptides and single alanine mutants of the full-length envelope confirmed the predominant role of Tys177 in mediating V2-V3 interaction. Of note, inhibition occurs on a broad range of HIV-1 isolates with different coreceptor tropism, highlighting the overall structural conservation of the coreceptor-binding site. In contrast, a CCR5 N-terminus-derived sulfated peptide inhibits selectively CCR5-tropic HIV-1 isolates. The molecular basis for this selectivity, which contrasts with the broad-spectrum activity of the V2-derived peptides, was mapped to a negatively charged residue next to the first sulfotyrosine (Tys10) of CCR5, which conflicts with a negatively charged residue frequently present at position 440 within the coreceptor-binding site of CXCR4-specific envelopes. These data confirm that the sulfated region of V2 is a functional mimic of the CCR5 N-terminus interacting with the coreceptor-binding site at the base of the V3 loop.